The regulation of protein synthesis by translational control RNA

Vol. 61, No. 2, 1974

THE

BIOCHEMICAL

REGULATION Ooris

OF

S.

PROTEIN

Kennedy,

Received

August

SYNTHESIS

Andre

Genetics University Storrs,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J.

BY TRANSLATIONAL

Bester”,

and

CONTROL

Stuart

M.

and Cell Biology Section, of Connecticut Connecticut 06268

RNA

Heywood

U-125

21,1974

SUMMARY The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [32P]-globin mRNA to 40s ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction. INTRODUCTION A low which

molecular

appears

to

translational the of

level

translation

from

synthesis and

myoglobin

of

these

report synthesis

suggest

40s

ribosomal

in

the

regulation

This

when

We have

tcRNA tcRNA

is

derived

with

the

a reticulocyte that

“Permanent address Tygerberg Hospital Cape Town South Africa

not from

and

and

partially

the

inhibits

its

subsequent

muscle the

415

inhibits

cell of

tcRNA. binding

utilization

of

type

as

inhibition The of

this

globin

blocking

the

the

inhibits

characterized

in

incorporation

:

the

translation

same

at

(tcRNA)

stimulate

effective

factors

synthesis RNA

tcRNA the

basis

tcRNA

Copyright 0 I974 by Academic Press, Inc. All rights of reproduction in any form reserved.

may

Muscle

mechanistic by

protein

and

inhibits

lysate

muscle

subunit

is

initiation

control

(1)

(1).

The

from of

isolated

reticulocytes

it

isolated

translational mRNAs

reticulocyte

concerned in

here

involved

mRNAs.

mRNAs is

been

(2).

and

while

has

heterologous

mRNA

muscle

RNA

(1,2).

of

homologous

RNA

be

weight

into

myosin

the

utilization

the

mRNA. of

results globin

both

This

protein

reported mRNA

polysomes.

to

the

Vol. 61, No. 2, 1974

Erythroblast tcRNA

tcRNA

decreases however,

results

in

suggest

that

reaction

in

an

tcRNA

The

the

label the

Each

tions

of

Leder tion

muscle

tcRNA

was

in

an

to

IEC

SB

the

tcRNA studies

mRNA

is

showed

at

binding

nevertheless

rotor.

as

labelled

globin

mRNA

was

and

translational

layering

the

the

buffer.

same

0.02M

A 3 ml, was were

416

the

method

The

using

chilled

onto

protein

(5).

done

45% for analyzed

sucrose 5 hrs

at by

PM

of

the the

(pH 10

to

Following

1.5

by

Tris-HCl

a 24 ml,

precipita-

(6).

analysis 1 ml

of

cetyl

and

to

prepara-

of

agent

volume

MgC12,

gradients

al

gradient the

sample

by

precipitating

Centrifugation The

was

rapidly

The

inhibition

et

RNA described

use.

measured

Anderson

unless

isolated

previously

to

is

30”

control

as

a 60%

density

0.003M

volume at

prior

radio-

Massachusetts)

incubation

prepared

for

The

done

ribosomes

bringing

(1).

final

was

the

conditions

normally

least

were

Sucrose

M KCl,

110

and

the

(Boston,

ml

activity

by

and

were

(3)

for

mixtures

0.1

gradient.

0.2

were

(CTAB)

after

containing

each

tested

polysomes

added.

and

each

a reticulocyte

muscle

that

Nuclear

[‘zP]

modified

performed

mercaptoethanol

of

as

reaction

containing

gradient

muscle

These

described

formation

bromide

were

of

complex

England

chickens

used

from

the

mixtures

in

blocking

lysates

reticulocytes

(4)

met-tRNA

cycloheximide

by

Incubations

was

Burtsztyn

incubation,

polysomes

addition

previously

New

added

and

trimethylammonium

bottom

as

Methionine-puromycin

of

buffer

were

treated

preparation

and

addition,

reaction,

Radioactive

muscle

synthesis.

In

formation.

reticulocyte

legends.

phenylhydrazine

(1).

rabbit

amount

figure

from

the

initiation

from

indicated.

(tcRNA)

to

synthesis

abortive

obtained and

the

otherwise

an

synthesis

was

in

binding

methionine-puromycin protein

of

isotope

stated

reaction.

METHODS

protein

active

met-tRNA

this

methionine-puromycin

preparation

cell-free

on

contradiction,

inhibits

of

AND

of

in

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

effect

apparent

increase

capable

MATERIALS

little

amount

resulting

still

from

has

the

lysate;

and

BIOCHEMICAL

incubation addition

7.4),

0.006M

30%

sucrose

cushion

was

25,000 continuous

of

RPM

at at

monitor-

the 5”

Vol.

61, No.

Table

2, 1974

1.

BIOCHEMICAL

The

Inhibition

of

AND

Globin

BIOPHYSICAL

Synthesis

by

RESEARCH

COMMUNICATIONS

tcRNA CPM

Additions

CPM

none 50

a-globin

91,065 uM

hemin

2 pg

muscle

Hemin 2 vg

130,045

30,340

tcRNA

+ muscl

e tcRNA

reticul

ocyte

31,245 86,455

tcRNA

%one “3

pg

muscle

462,860

257,840

203,960

36,480

11,730

23,850

tcRNA

Incubations were performed at 35” for 30 min. as Reactions contained 5 PC [35’S]-methionine except per reaction, Isolation and characterization of were as described by Dintzis (9). Al I radioactivity hot trichloroacetic acid precipitation.

ing

on

B-globin

a Gi 1 ford

scintil

lation

Spectrophotcnneter.

described in Methods. * which contained 20 uC c1- and f3-globin chains was determined after

Radioactivity

was

determined

by

liquid

counting.

RESULTS Previ synthesis is

known

ous

resu

of

hemog

t 0 effect

cell-free

amino

addition

of

protein

but

synthesis. tcRNA has on fore,

heme has Also,

is

stimulation to

determine

have

demonstrated

obin

(1)

vely

prolong

acid

in

increases no

the the

in

on

agreement in

of

the

of

with

globin

CI- and

B-chain

if

muscle

tcRNA

heme,

a compound

which

shown

tcRNA results

in

lysate

Table

to

1,

the into

inhibit

(I)

globin

reticylocyte

Bogdanovsky

reticulocyte

both

reticulocyte

incorporated

muscle

synthesis,

417

the

As

synthesis. of

inhibits

inhibit

of

previous

effect

can

radioactivity of

our

tcRNA

ability (7).

ability

of

added

initiating

amount

inhibiting

a differential

muscle

systems

the

effect

that absence

incorporating

ineffective

demonstrated the

ts

initiation It

was

of

interest,

d-

and

B-globin

et

al

(2)

factor

RNA there-

synthesis

BIOCHEMICAL

Vol. 61, No. 2, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

6

500

IO

IO

400

300

!O

200

I

100

4

500

40

400

80

300

*O

‘0 x

8

e 1 r

s f F 1 5 Q

200

B

P 5 8

; .a N

4

100

50(

140

40(

80

20

30( ,

8

20(

4

lO(

FRACTION

NUMBER

418

if 5 0 u

Vol.

61, No.

to

the

2, 1974

same

c1- and

degree

muscle

chain

tcRNA.

Bogdanovsky a-globin

et

al

Sucrose

reason

(2)

that

activly

labelled

subunit

as

as

reticulocyte the

portion

&OS of

polysome amino

into

the

tcRNA

is

there

into is

nascent

added

polysomes

a substantial

would

appear

entry

of

Figure

mRNA

chains

mRNA

1.

in that into

to

treated

of

to

the

reaction

to

the

40s

polysome

muscle ribosomal

tcRNA

region inhibits structures

the

as

similar.

On (Fig.

subunit

the

and

pro-

as

in

the

globin

synthesis

during

the

[3H]-

hand, the

binding

as

expected,

with

gradient.

1t

by

the thus

blocking of

of incor-

associated

initiation

when

subsequent Also,

sucrose

the

control

other

its

presence

of

both

radio-

associated

well

the

lB),

radioactivity the

in

both

inhibited.

of

the

incorporation of

or

ribosomal

a substantial

mixture

in

40s

control, fraction

completely

tcRNA

mRNA

polysomes

are

muscle

Al though,

the

unclear. containing

of

the

of

stimulates is

of

how-

finding

incubations,

absence

both

inhibition

the

32 PI-globin [

this

the

the

RNA

with

the

ribosomal

almost

the

addition,

lysate

decrease the

In chains

is

in

to

presence

polysomes.

compared found

or

1, tcRNA;

synthesis

sediment

less

gradient. nascent

to

slightly

is

tcRNA

[32P]-globin

poration

the the

reticulocyte

muscle

subunit

of

found

muscle

factor

lysate

In

Table

to

B-globin

1.

in

relationship

absence

into

is

radioactivity

region acids

there

ribosomal

the

the

incorporated

tcRNA

than

Figure is

its

COMMUNICATIONS

susceptible

reticulocyte in

shown by

initiation

of

mRNA

be

and

degree

in

As

more

reticulocyte

shown

globin

well

this

mRNA

RESEARCH

inhibited

somewhat

for

globin is

manner.

is

analysis

tcRNA

BIOPHYSICAL

substantially

a greater

gradient

reticulocyte

and

The

[32P]-labelled

with

are

to

AND

a differential

synthesis

synthesis

added

in

synthesis

e-globin

of

or

B-globin

ever, by

BIOCHEMICAL

the protein

The inhibition of globin mRNA binding to ribosomes by muscle The reactions were incubated for 5 min. at 30” with tcRNA. [32P]-globin mRNA and subsequently terminated and analyzed by sucrose density gradient centrifugation as described in Fractions were collected on Millipore filters, dried, Methods. B 2 ug muscle A, control, and the radioactivity determined. tcRNA, and C, 2 ug reticulocyte tcRNA added to incubation mixtures. 50 UM hemin was present in all reaction mixtures. H [32P]-globin mRNA, O--O [3H]-amino acid mixture (5 WC per reaction).

419

BIOCHEMICAL

Vol. 61, No. 2, 1974

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BOTTOM

Figure

2.

synthesis which mode

while the

of

TOP

The labelling of ribosomes with [ 35 S]-met-tRNA in the presence Reactions were incubated for 10 min. at and absence of tcRNA. 30” with 10 uC [35S]-methionine per sample. Analysis of the reactions was performed as described in Methods. 50 uM hemin was present in all reactions mixtures.. A, control having a total of 201,300 acid precipitable CPM after incubation, 6, 1 ug muscle tcRNA added with 68,000 acid precipitable CPM; C, 1 ug reticulocyte tcRNA added with 214,400 acid precipitable CPM.

mRNA action

reticulocyte binding of

tcRNA

tcRNA reaction is

not

is

is

ineffective.

inhibited

known.

420

The by

tcRNA

precise or

if

manner it

is

the

by only

Vol.

61, No.

In

2, 1974

order

various

BIOCHEMICAL

to

determine

ribosomal

were

sucrose

density

fractions between

the 2B),

it

peak,

a decrease

subunit

and

be

incubation

2C),

with

muscle

is

2A)

the

and

the

ribosomes

single

the

the

other

tcRNA

with

the

80s

the

binding

ing

with

the

of

met-tRNA.

This

possibility

(4).

As

shown

in

added

to

the

the

Although binding

an

capable

of

3,

to

is

the

the

with

that

tested

of

[35S]-met-tRNA is

present

in

found

results

S]-met-tRNA

is

added

obtained

that

complex the

amounts in

sedimenting

suggested

using

increase

the

globin

is

the

the

addition

met-tRNA

of

formed

muscle

amount

of

though contain-

reaction tcRNA

are

[5’S]-met-puromycin presumably

formation

bound

even

met-puromycin

synthesis

during upon

amount

35

ribosomal

inhibits

ribosomal

tcRNA

the

added

increasing

an

of

40s

[3SS]-met-tRNA

[

is

80s

ribosomes

show in

reacting

when

tcRNA

results

increase

an was

there

mRNA

these

actual

Figure

muscle

of

camp 1 ex,

inhibited

ribosome

the

reticulocyte

of

tcRNA

made tcRNA

80s

to

various

observed.

peak

muscle

the is

tcRNA

comparing

not

a radioactive

is

formed.

are

mixtures

the

muscle

when in

reaction of

a peak

less

subunit

control

mRNA

lysates

is

noted

when

in

the

containing

muscle

bound

when

in

lysates

a comparison

of

(SOS)

ribosomal

these

increase

appearance

there

differences

ribosomes of

the

Although 405

If

35 S]-met-tRNA

[

peptidyl-tRNA

containing

an

COMMUNICATIONS

precipitation 2.

is

of

of

reaction

there

amount

analysis

Figure

the

polysomes,

with

appearance

and

and

reticulocyte

subsequent

in

that

mixture.

The

the

RESEARCH

met-tRNA of

The

shown

seen

in

with

associated

out. and

(Fig.

can

the

associated

(Fig.

(6)

control

(Fig.

the

carried

CTAB

of

incubations

gradients

with

BIOPHYSICAL

presence

structures,

[ 35 S]-methionine on

the

AND

of

of

muscle

to

80s

by

the

blocking

initiation

tcRNA

there

ribosomes

is

which

is

puromycin.

DISCUSSION Translational to

inhibit

suggest initiation

the that

control utilization tcRNA

of

protein

RNA has of

inhibits synthesis.

been

different the

binding

shown

to

mRNAs

(1).

of

This

421

could

mRNA be

be

specific The

to

the

in studies

ability

reported

ribosome

accomplished

its

during by

a site

here the

Vol.

61, No.

2, 1974

BIOCHEMICAL

AND

05

BIOPHYSICAL

or

of near

then

AUG

Staehelin

in

eukaryotic

the

binding

Further

and of

latter

is

of

muscle

myosin

and

not

myoglobin

subsequent

likely

our

previous

an effective

to

et globin inhibitor

synthesis.

lve the

the

possibility

due

to

muscle mRNA

the

(2).

synthesis of

and globin

Recent

findings

422

tcRNA

may but

contain allowing

formation.

and

We have

a complex

binding

however,

specificity (1)

may reaction.

such

alternatives;

results al

complex

of

methionine-puromycin these

region

methionine-puromycin

blocking

test

were was from described

stranded

initiation

the

thereby

less

on

a double

Alternatively,

seems

tcRNA

of

suggested

required

Bogdanousky

forming

An abort

capable

are

of

effect

is

and

how

those

still

ribosomes

clear

to

tcRNA

to

mRNA

site.

have

not

relate

globin

systems.

met-tRNA

possibility It

but (8)

bind

experiments

to

binding

mRNA,

and

codon

tcRNA

ribosomal

lacking

occurring an

muscle

the

form,

Schrcier

tc RNA

Incubations The formation of [35S]-methionine-puromycin. 50 pM hemin for 5 min. at 30” as described in Methods. present in all reaction mixtures. The tcRNA was prepared Methionine-puromycin formation was measured as muscle. by Leder and Burtsztyn (4).

3.

recognition at

COMMUNICATIONS

2

I

pg Figure

RESEARCH

of those

studied

have synthesis from

reaction.

reported

here

that it

this

the

the

shown

the

inhibitory while

does laboratory

indeed

reticulocyte inhibit suggest

Vol.

that

61, No. 2, 1974

polysomal

polysomal inhibits

as

mRNA while the

elsewhere). reported

tcRNA

BIOCHEMICAL

preparations tcRNA of

Therefore,

the

Bogdanousky

from

these

of

tcRNA

al

from

non-ribosomal

non-functional

(2)

different

populations

are

This work recipient

was supported by NIH Grant of a NIH Career Development

RESEARCH

stimulate

stimulating et

BIOPHYSICAL

muscle

preparations

utilization

by

AND

mRNAs

activity may

be

present

of due

in

number Award,

the

the

to the

the

COMMUNICATIONS

translation bound

(to

be

of

mRNA

in

muscle

published

reticulocyte possibility

RNA that

cell.

HD 03316. number

S.M. Heywood GM 18904.

is

a

REFERENCES 1. 2.

2: 5. 60 7. 8. 9.

S. M., Kennedy, D. S., and Bester, A. J. (1974) Proc. Nat. Acad. Sci., U.S., 2, 2428-2431, (1973) Biochem. Bogdanousky, D., Hermann, W., and Schapira, G. Biophys. Res. Comm., 2, 25-32. Heywood, S. M. (1970) Proc. Nat. Acad. Sci., U.S., 67, 1782-1788. (1966) Biochem. Biophys. Res. Comm., 2, Leder, P. and Bursztyn, H. 233-245. (1972) Proc, Nat. Acad. Sci., U.S., Anderson, W. F. and Crystal, R. G. f&, 706-711. Hunt, T. and Jackson, R. J. (1972) Biochem. Biophys. Darnbrough, C., 4CJ 1556-1564. Res. Comm., Gross, M. and Rabinovitz, M. (1972) Proc. Nat. Acad. Sci., U.S.3 2, 1565-1571. (1973) Regulation of Transcription Schreier, M. H. and Staehelin, T. and Translation in Eukaryoties, pp* 335-349, Springer-Verlag, New York, (1961) Proc. Nat. Acad. Sci., U.S., 47, 247-261. Dintzis, H. M.

Heywood,

423